Cellular and molecular mechanisms of action of the novel adjuvant polyphosphazene

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Date

2013-01-23

Author

Awate, Sunita

Type

Thesis

Degree Level

Doctoral

Abstract

Adjuvants are critical components of modern vaccines. They are added to improve the host’s immune responses to the vaccine antigens. Understanding the mechanisms of action of adjuvants is critical in the rational design of vaccines. The novel adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) has shown great potential as a vaccine adjuvant, but the mechanisms that mediate its adjuvant activity have not been investigated. Hence, the present investigations were undertaken to understand the molecular and cellular mechanisms of action of PCEP. First, we investigated in vivo the capacity of PCEP to induce innate immune responses at the site of injection. PCEP induced time-dependent changes in the gene expression of various “adjuvant core response genes” including cytokines, chemokines, innate immune receptors and interferon-induced genes. We also observed that PCEP enhanced production of various cytokines including pro-inflammatory cytokines and chemokines such as CCL2, CCL4, CCL12 and CXCL10 locally at the injection site but no systemic responses.
Due to the potent chemotactic potential of local cytokines and chemokines produced post-injection of PCEP, we observed increased recruitment of various myeloid and lymphoid cells at the injection site. Neutrophils and macrophages were recruited in significantly higher numbers followed by monocytes and dendritic cells (DCs). In addition, there was increased recruitment of T and B lymphocytes at the injection site. Further, confocal studies revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells at the site of injection. Whole body in vivo imaging of mice injected intramuscularly with PCEP revealed localized distribution of PCEP post-injection in the muscle tissue. Approximately 70% of PCEP was cleared from the injection site within 24 h post-injection, but there was evidence of PCEP retention up to 12 weeks post-injection. Although we could not detect PCEP in the draining lymph nodes, we observed significant increase in neutrophil, macrophage, monocyte and DC numbers, with the latter cell population being most abundant.
We observed that in vivo PCEP upregulates NLRP3 gene and pro-inflammatory cytokine expression at the injection site. Since caspase-1 is a critical component of NLRP3 inflammasome and known to plays an important role in the release of IL-1β and IL-18, we examined the role of caspase-1 in PCEP-mediated secretion of IL-1β and IL-18 by splenic DCs. Pre-treatment of splenic DCs with the caspase-inhibitor YVAD-fmk significantly inhibited IL-1β and IL-18 secretion in response to PCEP. Although PCEP was taken up by the DCs, it failed to induce DC maturation (expression of MHC class II and co-stimulatory molecules CD86 and CD40). In addition, PCEP did not induce direct activation of naïve T cells. However, when naïve B cells were directly activated, PCEP induced significant production of IgM and IL-6. Further, immunization of mice with OVA plus PCEP significantly increased the production of OVA-specific IFN-γ by CD4+ T cells and CD8+ T cells suggesting that PCEP can generate antigen-specific T cell responses.
Taken together, these results suggest that adjuvant activity of PCEP depends on creating a strong immunocompetent environment at the site of injection by activating innate immune responses, which involves modulation of adjuvant core response genes, production of cytokines and chemokines, recruitment of various immune cells and presumably activation of inflammasomes. Together, all these mechanisms might contribute to the adjuvant activity of PCEP.